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Generation of Free Radical Intermediates from Foreign Compounds
by Neutrophil-derived Oxidants
B. Kalyanaraman and Peter G. Sohnle
National Biomedical ESR Center, Department of Radiology, and Infectious Diseases Section, Department ofMedicine, The Medical
College of Wisconsin, Milwaukee, Wisconsin 53226; and the Research Service, Veterans Administration Medical Center, Wood,
Wisconsin 53193
Abstract
A large number of foreign compounds, including many drugs,
industrial pollutants, and environmental chemicals, can be
oxidized under appropriate conditions to potentially toxic free
radical intermediates. We evaluated the ability of the oxidants
produced by the neutrophil myeloperoxidase system to generate
free radical intermediates from several such compounds. Sodium
hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine
chloramine were both found to be capable of producing free
radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible
light spectroscopy and by direct electron spin resonance (for
the chlorpromazine and aminopyrine radicals) or by spintrapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those
present in the supernatants of stimulated neutrophils in the
absence of added taurine) also were found to be capable of
generating free radical intermediates. The production of the
oxidants and the ability of neutrophil supernatants to generate
these radicals were almost completely eliminated by sodium
azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent
a new metabolic pathway for these substances and could be
important in the processes of drug toxicity and chemical
carcinogenesis.
Introduction
Many chemical compounds can be converted into free radical
forms. The latter species are usually quite reactive and shortlived because they have a single unpaired electron in their
outer orbital. Recently, evidence has accumulated to indicate
that free radical intermediates may be important in the toxicity
of a large number of substances (1-4). Free radicals can be
formed by several mechanisms, including one-electron oxidation, one-electron reduction, homolytic cleavage of a C-H
bond, and in some cases by two-electron oxidation/reduction
reactions. Enzyme-catalyzed oxidation also provides a mechanism by which free radical intermediates can be formed.
Examples of this process include the generation of the chlorpromazine free radical cation (CPZ +)' by hydrogen peroxide
Address reprint requests to Dr. Sohnle, Research Service/ 151, Veterans
Administration Medical Center, Wood, WI 53193.
Received for publication I October 1984 and in revised form 3
January 1985.
1. Abbreviations used in this paper: AP +, aminopyrine free radical
cation; CPZ +, chlorpromazine free radical cation; DMPO, 5,5-di-
The Journal of Clinical Investigation, Inc.
Volume 75, May 1985, 1618-1622
1618
B. Kalyanaraman and P. G. Sohnle
and horseradish peroxidase (5) and the prostaglandin-synthetasedependent production of the aminopyrine free radical cation
(AP. +) by a one-electron oxidation of aminopyrine (6). However, a two-electron oxidative pathway seems to generate free
radicals in the tyrosinase-catalyzed oxidation of catecholamines (7).
Oxygen-containing free radicals such as the superoxide
anion and the hydroxyl radical can be generated by several
mechanisms in living organisms, including the oxidative microbicidal processes of phagocytic cells (8). Recently, neutrophils
have been shown to produce genetic mutations in bacteria (9)
and cytogenetic damage to the chromosomes of Chinese
hamster ovary cells (10), apparently through the production
of the hydroxyl radical. There is a significant correlation
between the development of local cancers and the presence of
infectious or noninfectious inflammatory processes (11). The
possibility exists that neutrophils at inflammatory sites can
directly cause mutations in surrounding cells, leading to the
development of cancers. However, stimulated neutrophils also
produce powerful oxidants through their myeloperoxidase system, which has the capacity to release hypochlorous acid into
the surrounding medium (12, 13). Therefore, an alternative
explanation for the association of inflammation and cancer is
that neutrophils can oxidize potential carcinogens such as
environmental pollutants, drugs, or even certain chemical
compounds in foodstuffs (14) to highly toxic electrophilic free
radical forms that might result in the neoplastic transformation
of surrounding cells. This possibility is also consistent with the
existing hypothesis that the ultimate metabolites initiating
carcinogenesis are electrophiles (15). In addition, a similar
mechanism might be involved in the toxicity of certain drugs
that can be oxidized to free radical forms.
This study was undertaken to determine if the oxidants
produced by neutrophils could generate potentially toxic free
radical intermediates from certain foreign compounds. We
report that hypochlorous acid, taurine chloramine, and the
oxidants produced by stimulated neutrophils can convert
chlorpromazine and aminopyrine to their free radical cation
intermediates (CPZ. + and AP +) and can also generate the
phenyl radical (Ph .) from phenylhydrazine.
Methods
Chemicals. Aminopyrine, chlorpromazine, phenylhydrazine, taurine,
zymosan, sodium azide, potassium iodide, and catalase were obtained
from Sigma Chemical Company, St. Louis, MO. Sodium hypochlorite,
obtained from Aldrich Chemical Company, Milwaukee, WI, was used
as a source of hypochlorous acid/hypochlorite ion (HOCI/OCI-) solutions and was quantitated by its absorbance at 292 nm. 5,5-dimethylI-pyrroline N-oxide (DMPO), obtained from Aldrich Chemical Co.,
was purified over activated charcoal and kept frozen under nitrogen.
methyl- l-pyrroline N-oxide; ESR, electron spin resonance; HOCI/
OC1-, hypochlorous acid/hypochlorite ion; Ph., phenyl radical.
Downloaded from http://www.jci.org on June 16, 2017. https://doi.org/10.1172/JCI111868
Phenylhydrazine was prepared in 0.2 M acetic acid immediately before
Results
use to minimize autoxidation. Zymosan was opsonized by incubation
of 5 mg in 1 ml of fresh human serum for 30 min at 370C followed
Production offree radicals by sodium hypochlorite and taurine
by washing three times in saline.
chloramine. Because stimulated neutrophils have been shown
Cells. Heparinized peripheral blood from normal human volunteers
to release hypochlorous acid (17) and stable chloramines (18)
was subjected to Hypaque-Ficoll density gradient centrifugation with
into the surrounding medium, we first evaluated the capacity
the isolation of neutrophils as previously described (16). Red blood
of these substances to oxidize foreign compounds to their free
cells were removed by hypotonic lysis. Resulting preparations consisted
radical intermediates. Both sodium hypochlorite solutions and
of 98-99% neutrophils of >96% viability (by Trypan blue dye exclusion)
the stable chloramine, taurine chloramine, were able to produce
with little or no erythrocyte contamination.
the free radical intermediates from chlorpromazine, aminoAssays. The release of HOCI/OCl- into the medium was quantitated
pyrine, and phenylhydrazine as detected using ESR and/or
using a modification of the chlorination of taurine method (17). This
visible light spectroscopy. Fig. 1 shows the ESR spectra of
assay uses the ability of taurine to react with HOCl/OCI- to form
taurine chloramine, which is an oxidant more stable than HOCI/OC1CPZ. + and AP- + generated by taurine chloramine (300 MM)
itself. The neutrophils (5 X 106/ml) were incubated in phosphate
and the DMPO-phenyl adduct generated by sodium hypochlobuffered saline containing 5 mM potassium chloride, 1 mM calcium
rite (400 MM). Similar spectra were obtained for CPZ- + and
chloride, 1 mM magnesium sulfate, and 5 mM glucose at 7.4. In most
AP- + generated using HOCl/OCl- and the DMPO-phenyl
instances, taurine was added before incubation and opsonized zymosan
adduct generated using taurine chloramine (data not shown).
(2 mg/ml) was used as the neutrophil stimulant. Incubations were
The
ESR spectra obtained for CPZ + and AP- + were quite
generally carried out at 22°C. (Previous experiments in our laboratory
to those described in previous studies (5, 6), and
comparable
have demonstrated optimal release of HOCl/OCI- at 220C with the
the ESR parameters of the DMPO-phenyl adduct agreed with
use of opsonized zymosan as the stimulus.) In some experiments,
the values in the literature (22).
sodium azide (10-4 M) was used to inhibit neutrophil myeloperoxidase.
After incubation, the tubes were centrifuged and the supernatants were
The colored, relatively stable CPZ- + and AP - + radicals
removed for quantitation of taurine chloramine. The latter was detercould be quantitated spectrophotometrically. However, whereas
mined using the oxidation of iodide (added as 20 mM potassium
CPZ- + appeared immediately upon addition of the oxidants,
iodide) to iodine, which could be detected spectrophotometrically in
AP - + required -60 s to develop. Both radicals began to
an excess of I- as I- at 350 nm. Catalase (100 jig/ml) was added before
disappear within several minutes. Fig. 2 shows the quantity of
the KI in this assay to destroy any remaining hydrogen peroxide,
+ and AP + produced by varying amounts of taurine
CPZ
which can (slowly) oxidize iodide to iodine. The data were expressed
chloramine.
Note that detectable quantities of CPZ + were
as nanomoles of HOCI/OCI- produced by 5 X 106 neutrophils using
as little as 10-30 juM taurine chloramine, an
produced
by
a standard curve of sodium hypochlorite concentrations. Generation
amount well within the capacity of neutrophils to produce (see
of stable oxidants produced by neutrophils, as recently described by
below). In addition, Fig. 2 A also shows that there was a linear
Weiss et al. (18), was estimated in a similar manner except that taurine
was not added to the incubation medium.
relationship between CPZ- + and the quantity of taurine
Generation of free radical intermediates from chlorpromazine,
chloramine present. Quantitation of the amount of CPZ +
aminopyrine, and phenylhydrazine was studied using as oxidants
and AP- + produced by HOCl/OCl- was more difficult because
sodium hypochlorite, taurine chloramine (which was produced by
adding sodium hypochlorite to an excess of taurine) (17), and supernatants of stimulated neutrophils. The conditions were similar to those
CPZt CH2CH2CH2N(CH3)2
used in the assay for HOCI/OCI- except that some of the experiments
involving chlorpromazine were carried out at 37°C and using a reduced
concentration (5 mM) of taurine (preliminary experiments suggested
that the higher taurine concentration might be somewhat inhibitory of
CPZ. + generation). The free radical cations of chlorpromazine and
aminopyrine are both relatively stable at pH 5.0, and both absorb light
+
in the visible range. Therefore, the pH of the solutions containing
Cj-Ph AP-._
chlorpromazine and aminopyrine was first adjusted to pH 5.0 using
-CH3
acetic acid. CPZ. + was determined spectrophotometrically at 526 nm
i20G
CH3-I
CH3
using an extinction coefficient of 1.07 X I04 M- cm-' (19) and AP +
at 480 nm using an extinction coefficient of 2.5 X 103 M-' cm-' (6).
Direct electron spin resonance (ESR) spectroscopy was used to
confirm the presence of CPZ * + and AP +, whereas production of Ph .
was quantitated using ESR spin trapping techniques (20, 21). The
samples were transferred to a miniature quartz ESR flat cell that was
placed in a dewar contained in the cavity of the ESR spectrometer
(Varian E-109, Varian Associates, Inc., Palo Alto, CA). The ESR
spectra were generated at room temperature. Spin-trapping of the
phenyl radical by DMPO and subsequent evaluation of the DMPOphenyl adduct by ESR was used to detect generation of this radical
from phenylhydrazine by the oxidants described above. These studies
Figure 1. Electron spin resonance spectra of free radical intermediates generated by HOCI/OCI- or taurine chloramine. Top, chlorwere carried out in PBS at pH 7.4 after adding 35 mM DMPO and
10 mM phenylhydrazine to solutions of sodium hypochlorite, taurine
promazine free radical cation (CPZ +) produced by 300 AM NaOCI
chloramine, or the neutrophil supernatants. These samples were kept
plus 20 mM taurine. Middle, Aminopyrine free radical cation (AP. +)
frozen until they were analyzed by being thawed, transferred to the
produced by 300 MM NaOCI plus 20 mM taurine. Bottom, spin
adduct of the phenyl radical (Ph- ) produced by 400 MM NaOCI in
miniature flat cell, and investigated by ESR as described above. Spectra
the presence of the spin trap, DMPO (35 mM). Structures of the free
obtained in these studies were compared with known ESR spectra for
radicals are shown to the right of their respective spectra.
CPZ * +, APT +, and the DMPO-phenyl adduct (5, 6, 22).
I
ci
/V
-
-
Neutrophils and Free Radicals
1619
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A
'D
200
D
N
CD
0+.100
P
C)
0
0
20 40 60 80- 100
pM NaOCI plus 5 mM Taurine
100
80
'a
0
d.0c
0)
60
40
20
o
200 400 600 800 1000
pM NaOCI plus 5 mM Taurine
Figure 2. (A) Generation of
the chlorpromazine free
radical cation (CPZ I) by
taurine chloramine. Chlorpromazine (10 mM) was
added to varying concentrations of NaOCI in the
presence of 5 mM taurine
at pH 5.0. Optical density
was determined at 526 nm
immediately after addition
of chlorpromazine. (B)
Generation of the aminopyrine free radical cation
(AP I) by taurine chloramine. Aminopyrine (10
mM) was added to varying
concentrations of NaOCI in
the presence of 5 mM taurine at pH 5.0. Optical
density was determined at
480 nm when the color
reached its maximal inten-
sity.
the radicals produced seemed to decay more rapidly than
when taurine chloramine was used as the oxidant.
Production offree radicals by neutrophil-derived oxidants.
Neutrophils were stimulated with opsonized zymosan in the
presence or absence of taurine and the supernatants were
tested to determine the amount of HOCl/OCl- that had been
produced and their ability to generate the free radical intermediates. Table I shows that the stimulated neutrophils released
into the medium 88.3±23.6 nMol of HOCl/OCl- per 5 X 106
cells. As an estimate of the amount of stable oxidant produced,
5 X 106 stimulated neutrophils produced an equivalent of
12.3±0.8 nmol of HOCl/OCl- in the absence of taurine.
Significant amounts of CPZ. + and AP. + were detected spectrophotometrically when 10 mM of either chlorpromazine or
aminopyrine were introduced into these supernatants after
adjusting the pH to 5.0 to increase the stability of the radicals.
These data are shown in Table II for chlorpromazine and
Table III for aminopyrine. Note that reduction of the pH was
done to increase stability of the radicals so they could be
measured; generation of these species apparently occurs at
Table L Production of Hypochlorous Acid by Neutrophils
Table II. Generation of CPZ. + by Neutrophil
Supernatants as Determined Spectrophotometrically
Additions
nmol CPZ * + per 5 X 10'
cells in 1.0-ml cultures
Part A. 220C, 2-h incubation
Cells alone
Cells plus taurine*
Cells plus op zyt
Cells plus taurine plus op zy
Cells plus taurine plus op zy plus azide§
0±0
0±0
2.5±0.7
68.1±9.9
0.1±0.1
Part B. 370C, 2-h incubation
Cells alone
Cells plus taurine
Cells plus op zy
Cells plus taurine plus op zy
Cells plus taurine plus op zy plus azide
1.5±0.8
1.9±0.5
10.4±2.4
80.7±13.3
2.1±0.5
Part C. 370C, varying incubation periods
Cells plus taurine, 120 min
Cells plus taurine plus op zy, 5 min
Cells plus taurine plus op zy, 20 min
Cells plus taurine plus op zy, 60 min
Cells plus taurine plus op zy, 120 min
1.4±0.8
19.2±4.3
41.6±2.8
44.1±7.6
78.8±18.6
* Taurine concentration was 20 mM for experiments at 22°C, 5 mM
for experiments at 37°C.
t op zy, opsonized zymosan (2 mg/ml).
§ Azide concentration was 0.1 mM.
Data represent mean±SE for three experiments (parts A and C) or
four experiments (part B). Statistical significance (unpaired t test):
Part A, cells vs. cells plus op zy, P < 0.05; cells plus taurine vs. cells
plus taurine plus op zy, P < 0.01; part B, cells vs. cells plus op zy, P
< 0.02; cells plus taurine vs. cells plus taurine plus op zy, P < 0.01.
physiological pH's (5, 6). Table II also demonstrates the
production of CPZ. + by the supernatants of neutrophils
stimulated (at 37°C) at various times beforehand. The ability
to generate this radical began at or before 5 min of incubation
and increased over the 2-h incubation period. Generation of
Ph- from phenylhydrazine was detected by ESR-spin trapping
Table III. Generation of AP. + by Neutrophil
Supernatants as Determined Spectrophotometrically
Additions
nmol HOCi/OCI- per 5 X 106
cells in 1.0-ml cultures
Additions
nmol AP-' per 5 X 10'
cells in 1.0-ml cultures
Cells alone
Cells plus taurine*
Cells plus op zyt
Cells plus taurine plus op zy
Cells plus taurine plus op zy plus azide§
5.8±1.3
7.0±0.9
12.3±0.7
88.3±23.6
8.7±0.6
Cells alone
Cells plus taurine*
Cells plus op zyt
Cells plus taurine plus op zy
Cells plus taurine plus op zy plus azide§
0.2±0.1
0.7±0.1
0.8±0.4
9.5±0.2
0.5±0.1
* Taurine concentration was 20 mM.
t op zy, opsonized zymosan (2 mg/ml).
§ Azide concentration, 0.1 mM.
Data represent mean±SE for three experiments carried out at 22°C
and a 2-h incubation. Statistical significance (unpaired t test): cells
alone vs. cells plus op zy, P < 0.05; cells plus taurine vs. cells plus
taurine plus op zy, P < 0.05.
1620
B. Kalyanaraman and P. G. Sohnle
* Taurine concentration was 20 mM.
* op zy, opsonized zymosan (2 mg/ml).
§ Azide concentration was 0.1 mM.
Data represent mean±SE for three experiments carried out at 22°C
and a 2-h incubation. Statistical significance (unpaired t test): cells
alone vs. cells plus op zy, NS; cells plus taurine vs. cells plus taurine
plus op zy, P < 0.001.
Downloaded from http://www.jci.org on June 16, 2017. https://doi.org/10.1172/JCI111868
at pH 7.4 in the neutrophil supernatants when 35 mM DMPO
and 10 mM phenyihydrazine were added (Table IV). Sodium
azide (an inhibitor of myeloperoxidase) suppressed production
of hypochlorous acid and the ability of the supernatants to
generate all three radicals. Production of the free radicals in
each case was also reduced in the absence of taurine.
Discussion
This study shows that the type and quantity of oxidants
produced by stimulated human neutrophils can generate free
radical intermediates from chlorpromazine, aminopyrine, and
phenylhydrazine. As an integral part of their microbicidal
mechanisms, stimulated neutrophils remove from the medium
relatively large quantities of oxygen, which are sequentially
converted to the superoxide anion radical, hydrogen peroxide,
and HOCl/OCl- (23). In contrast to most other peroxidases,
myeloperoxidase converts hydrogen peroxide and chloride ion
to a strong oxidant that can diffuse away from the enzyme
complex (and into the medium surrounding the cell) as HOCI/
OC- (13). The latter is an effective microbicidal agent that
readily oxidizes many biologic molecules and can react with
amino groups to form chloramines of varying stability (24).
Since in the presence of neutrophils, HOCl/OCI- is a shortlived species, we found it necessary to trap this compound as
taurine chloramine, a molecule that retains significant oxidizing
capacity, to show most clearly the ability of neutrophil supernatants to generate these free radical intermediates. Neutrophils
also seem to produce some of their oxidants in a stable form,
probably representing chloramines (18). With chlorpromazine
and phenylhydrazine, some activity of the supernatants was
present in the absence of taurine, presumably due to the
presence of these stable chloramines generated by the stimulated
cells. Alternatively, the combination of hydrogen peroxide and
myeloperoxidase released from stimulated neutrophils might
also have been capable of generating these free radical intermediates. In addition, supernatants from neutrophils triggered
in the presence of azide were still able to produce relatively
large amounts of the DMPO-phenyl adduct. One possible
explanation for this observation could be that hydrogen peroxide
might accumulate in the supernatants under these conditions
and could oxidize phenylhydrazine to Ph- in the presence of
Table IV Generation of Ph from Phenyihydrazine by
Neutrophil Supernatants as Measured by ESR-Spin-Trapping
-
Additions
Concentration of DMPO-Phin arbitrary units
Cells alone
Cells plus taurine*
Cells plus op zyt
Cells plus taurine plus op zy
Cells plus taurine plus op zy plus azide§
4.0±1.0
8.8±2.1
18.7±1.2
27.7±5.2
14.7±3.4
* Taurine concentration was 20 mM.
* op zy, opsonized zymosan (2 mg/ml).
§ Azide concentration was 0.1 mM.
Data represent mean±SE for three experiments using the supernatants generated from 5 X 106 cells/ml at 22°C and a 2-h incubation.
Statistical significance (unpaired t test): cells alone vs. cells plus op
zy, P < 0.001; cells plus taurine vs. cells plus taurine plus op zy,
P < 0.05.
certain metal ions, particularly ferrous iron. In summary, the
results of these studies demonstrate that HOCl/OCI- solutions,
taurine chloramine, and neutrophil oxidants in the form of
either taurine chloramine or in some cases, the stable oxidants,
can generate free radical intermediates from structurally diverse
foreign compounds.
A wide variety of compounds can be converted to free
radical intermediates by a number of oxidizing conditions,
including horseradish peroxidase and hydrogen peroxide, prostaglandin synthetase/arachidonic acid, ceruloplasmin, and the
microsomal cytochrome P450 system (3). The types of oxidants
produced by neutrophils also seem to be capable of this
conversion. The linear relationship that we observed between
the amount of CPZ- + generated and the quantity of taurine
chloramine added as expressed arithmetically (Fig. 2) suggests
that a two-electron oxidation process may have been involved
in this process. One possibility is that CPZ- + and AP- +
produced by HOCI/OCl- or taurine chloramine could have
been generated via a back reaction between the two-electron
oxidation intermediates and their respective parent compounds.
Under other conditions, Ph- seems to be produced from
phenylhydrazine by decomposition of phenyl diazene, the twoelectron oxidation intermediate (20, 25), and this mechanism
could be involved in the production of this species by the
neutrophil-derived oxidants.
The three compounds used in this study all have significant
in vivo toxicity under the appropriate conditions. Whereas
chlorpromazine is a safe and effective drug in most instances,
it does exhibit a variety of toxic effects. Both chlorpromazine
and aminopyrine can produce neutropenia when used therapeutically in humans (26, 27). This side effect has limited the
usefulness of aminopyrine as an antipyretic. The formation of
CPZ* + has been suggested as a cause of the toxic side effects
of chlorpromazine therapy by the interaction of this radical
with endogenous cellular reductants (28). The hydrazines are
an interesting group of compounds that have toxic and carcinogenic properties (25, 29). Covalent binding to the heme
moiety of hemoglobin by Ph. generated from phenylhydrazine
has been implicated in the toxicity of this compound for
erythrocytes (25). Under the proper conditions, neutrophils
might be able to oxidize other chemical compounds to their
free radical intermediates, and it is possible that this mechanism
could be involved in the toxicity of some of these compounds.
Local cancers often occur at sites of infectious or noninfectious inflammatory processes. Examples are squamous cell
carcinomas associated with the fistulas from chronic osteomyelitis (30), bladder carcinoma in schistosomiasis (31), testicular carcinoma in mumps orchitis (32), and gastrointestinal
carcinoma in inflammatory bowel disease (33). A number of
plausible explanations for this association between inflammation
and cancer are possible, including an increased rate of epithelial
turnover in the inflamed tissue, direct effects on cellular DNA
by a viral agent causing the inflammation, and chromosomal
damage caused by oxygen-containing free radicals produced
by the stimulated inflammatory cells. There is a reasonable
likelihood that many tissues in the human body are frequently
exposed to a wide variety of compounds that can be oxidized
to potentially toxic free radical intermediates. Sources of such
compounds may be exogenous, as in the cases of industrial
pollutants, drugs, and naturally occurring chemicals in foods;
or may be endogenous, as with mediators such as catecholamines. While some of the free radical intermediates may be
Neutrophils and Free Radicals
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stable enough to travel away from the site of generation, most
would probably act locally. Therefore, conversion of chemical
compounds to toxic-free radical intermediates by stimulated
phagocytic cells in a localized inflammatory process could be
an additional explanation for the association between inflammation and cancer.
If stimulated neutrophils can indeed oxidize various chemical compounds to their electrophilic free radical intermediates
in vivo, this process may represent a new pathway for the
metabolism of these compounds. This pathway could be
important in the toxicity of certain drugs and in the carcinogenic
potential of other chemical compounds.
Acknowledgments
The authors thank Miss Victoria Gollish for her excellent editorial
assistance.
This work was supported by the Veterans Administration Medical
Research Service and grants GM-29035 and RR-01008 from the
National Institutes of Health.
References
1. Docampo, R., and S. N. J. Moreno. 1984. Free radical metabolites
in the mode of action of chemotherapeutic agents and phagocytic cells
on Trypanosoma cruzi. Rev. Infect. Dis. 6:223-228.
2. Trush, M. A., E. G. Mimwaugh, and T. E. Gram. 1982.
Activation of pharmacologic agents to radical intermediates. Implications
for the role of free radicals in drug action and toxicity. Biochem.
Pharmacol. 47:412-426.
3. Mason, R. P. 1979. Free radical metabolites of foreign compounds
and their toxicological significance. In Reviews in Biochemical Toxicology, Vol. 1. E. Hodgson, J. R. Bend, and R. M. Philpot, editors.
Elsevier-North Holland, New York. 151-200.
4. Ts'o, P. 0. P., W. J. Caspary, and R. J. Lorentzen. 1977. The
involvement of free radicals in chemical carcinogenesis. In Free
Radicals in Biology, Vol. III. W. A. Pryor, editor. Academic Press,
New York. 268-275.
5. Piette, L. H., G. Bulow, and I. Yamazaki. 1964. Electronparamagnetic-resonance studies of the chlorpromazine free radical
formed during enzymatic oxidation by peroxidase-hydrogen peroxide.
Biochim. Biophys. Acta 88:120-129.
6. Lasker, J. M., K. Sivarajah, R. P. Mason, B. Kalanaraman,
M. B. Abou-Donia, and T. E. Eling. 1981. A free radical mechanism
of prostaglandin synthetase-dependent aminopyrine demethylation. J.
Biot. Chem. 256:7764-7767.
7. Mason, H. S. 1955. Comparative biochemistry of the phenolase
complex. Adv. Enzymol. Relat. Areas Mol. Biol. 16:105-184.
8. Leibovitz, B. E., and B. V. Siegel. 1980. Aspects of free radical
reactions in biological systems: aging. J. Gerontol. 35:45-56.
9. Weitzman, S. A., and T. P. Stossel. 1981. Mutation caused by
human phagocytes. Science (Wash. DC). 212:546-547.
10. Weitberg, A. B., S. A. Weitzman, M. Destrempes, S. A. Latt,
and T. P. Stossel. 1983. Stimulated human phagocytes produce cytogenetic changes in cultured mammalian cells. N. Engl. J. Med. 308:
21-30.
11. Templeton, A. C. 1975. Acquired diseases, Chapter 4. In
Persons at High Risk of Cancer. An Approach to Cancer Etiology and
Control. J. F. Fraumeni, Jr., editor. Academic Press, New York. 6984.
12. Babior, B. M. 1984. The respiratory burst of phagocytes. J.
Clin. Invest. 73:599-601.
13. Harrison, J. E., and J. Schultz. 1976. Studies on the chlorinating
activity of myeloperoxidase. J. Biot. Chem. 251:1371-1374.
14. Ames, B. N. 1983. Dietary carcinogens and anticarcinogens.
1622
B. Kalyanaraman and P. G. Sohnle
Oxygen radicals and degenerative diseases. Science (Wash. DC). 221:
1256-1263.
15. Miller, E. C., and J. A. Miller. 1981. Searches for ultimate
chemical carcinogens and their reactions with cellular macromolecules.
Cancer (Phila.). 10:2327-2345.
16. Sohnle, P. G., and C. Collins-Lech. 1978. Cell-mediated
immunity to Pityrosporum orbiculare in tinea versicolor. J. Clin.
Invest. 62:45-53.
17. Weiss, S. J., R. Klein, A. Slivka, and M. Wei. 1982. Chlorination
of taurine by human neutrophils: evidence for hypochlorous acid
generation. J. Clin. Invest. 70:598-607.
18. Weiss, S. J., M. B. Lampert, and S. T. Test. 1983. Long-lived
oxidants generated by human neutrophils: characterization and bioactivity. Science (Wash. DC). 222:625-626.
19. Pelizzetti, E., D. Meisel, W. A. Mulac, and P. Neta. 1979. On
the electron transfer from ascorbic acid to various phenothiazine
radicals. J. Am. Chem. Soc. 101:6954-6959.
20. Kalyanaraman, B. 1982. Detection of toxic free radicals in
biology and medicine. In Reviews in Biochemical Toxicology. Vol. 4.
C. E. Hodgson, J. R. Bend, and R. M. Philpot, editors. Elsevier/North
Holland, New York. 73-139.
21. Janzen, E. G. 1980. A critical review of spin-trapping in
biological systems. In Free Radicals in Biology. Vol. 4. W. A. Pryor,
editor. Academic Press, New York. 115-154.
22. Augusto, O., K. L. Kunze, and P. R. Ortiz de Montellano.
1982. N-phenylprotoporphyrin IX formation in the hemoglobin-phenylhydrazine reaction: evidence for a protein-stabilized iron-phenyl
intermediate. J. Biol. Chem. 257:6231-6241.
23. Root, R. K., and M. S. Cohen. 1981. The microbicidal
mechanisms of human neutrophils and eosinophils. Rev. Infect. Dis.
3:565-598.
24. Thomas, E. L. 1979. Myeloperoxidase, hydrogen peroxide,
chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial
components in bactericidal action against Escherichia coli. Infect.
Immun. 23:522-531.
25. Maloney, S. J., and R. A. Prough. 1984. Biochemical toxicology
of hydrazines. In Reviews in Biochemical Toxicology. Vol. 5. C. E.
Hodgson, S. R. Bend, and R. M. Philpot, editors. Elsevier/North
Holland, Amsterdam. 313-348.
26. Baldessarini, R. J. 1980. Drugs and the treatment of psychiatric
disorders. In The Pharmacological Basis of Therapeutics. 6th Edition.
A. G. Gilman, L. S. Goodman, and A. Gilman, editors. MacMillan
Publishing Co., Inc., New York. 391-447.
27. Flower, R. J., S. Moncada, and J. R. Vane. 1980. Analgesicantipyretics and anti-inflammatory agents: drugs employed in the
treatment of gout. In The Pharmacological Basis of Therapeutics. 6th
Edition. A. G. Gilman, L. S. Goodman, and A. Gilman, editors.
MacMillan Publishing Co., Inc., New York. 701.
28. Mayausky, J. S., H. Y. Cheng, P. H. Sackett, and R. L.
McCreery. 1982. Spectroelectrochemical examination of the reactions
of chlorpromazine cation radical with physiological macromolecules.
In Advances in Chemistry Series, No. 201. American Chemical Society.
443-456.
29. Toth, B. 1975. Synthetic and naturally-occurring hydrazines as
possible cancer causative agents. Cancer Res. 35:3693-3697.
30. Manale, B. L., and T. D. Brower. 1973. The significance of
bacterial flora in carcinoma in chronic osteomyelitis. Surg. Gynecol.
Obstet. 136:63-64.
31. Kuntz, R. E., A. M. Cheever, and B. J. Myers. 1972. Proliferative
epithelial lesions of the urinary bladder of non-human primates
infected with Schistosoma hematobium. J. Natl. Cancer Inst. 68:223235.
32. Gilbert, J. B. 1944. Tumors of the testis following mumps
orchitis. J. Urol. 51:296-300.
33. Saeed, W., S. Kim, and B. H. Burch. 1974. Development of
carcinoma in regional enteritis. Arch. Surg. 108:376-379.